The molecular analysis of cancer and genetic disorders that predispose to cancer has been invaluable in the discovery of genes and proteins that regulate normal biological processes and cellular pathways that participate in DNA repair, cell growth and differentiation. One such genetic disorder is adenomatous polyposis coli or APC. We mapped and then isolated the APC gene by positional cloning from a region of chromosome 5q. This gene is thought to be a tumor suppressor based on the genetics of the clinical disorder and the detection of somatic mutations in colorectal tumors. Mutations of APC may be critical and rate-limiting events in the development of colorectal tumors and very possibly other types of tumors. Therefore, understanding the role of the APC gene in the suppression of tumorigenicity is an important goal for basic biological and medical research. Our preliminary work has shown that transfer of normal, full-length APC into colorectal carcinoma cells in vitro alters morphology reduces cloning efficiency of the cells in soft agar and suppresses tumorigenicity. Here, we propose to extend this early work by defining functional domains of the APC protein and learning more about APC gene structure, alternative splicing and expression in various tissues. First, normal, full-length APC will be introduced into genetically characterized colorectal carcinoma cell lines, to determine more fully the phenotypic consequences of introducing APC. Next, in order to identify the separate functional domains of APC protein and their association with particular cellular phenotypes, the gene will be subdivided and transfected in shorter segments that represent the putative functional domains of the protein based on amino acid sequence. A similar approach will be undertaken for each of the alternative splice- forms of APC specific to colonic epithelial cells, postulating that each of these splice-forms encodes a variant APC protein of unique function. Lastly, inherited mutations observed in APC patients will be used to design constructs for gene transfer that may reflect other functional domains of the gene or protein, as the positions of different APC mutations are now known to be associated with unique clinical phenotypes. It is hoped that such studies will provide new insights into the biological function of this tumor suppressor gene and more generally into the ways in which normal cells can progress into neoplasia. This information may well guide the design for novel types of therapeutic interventions for tumors, i.e. gene therapy, and define potential cellular targets for pharmacological intervention.